Pen strep neo antibiotics

All cell culture was performed in HEPA-filtered cell culture cabinets. All media components were bought sterile or 0.22 μm filtered before use. Cells were grown at 37 °C in 5% CO₂. Cell density and viability were monitored with a 1:1 mix of cell culture medium and 0.4% Trypan blue. Stained cells were analyzed with a Countess II automated cell counter (ThermoFisher). Cells were independently tested negative for mycoplasma (Human Immunology Unit, WIMM). Jurkat T cells and derived cell lines were cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) HEPES buffer, and 1% (v/v) pen/strep/neo antibiotics (Sigma; complete RPMI). Cells were maintained between 0.1–1 × 10⁶ ml⁻¹. Human embryonic kidney 293T (HEK-293T) cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (v/v) FCS, 1% (v/v) pen/strep/neo antibiotics (Sigma), and 1% (v/v) glutamine (complete DMEM). The catalog numbers for commercial items can be found in Supplementary Table 1.